In the life cycle of HIV, the Rev protein regulates the temporal switch from the early regulatory to the late lytic phase by binding to a highly structured RRE (Rev Responsive Element) RNA. Two questions regarding Rev:RRE RNA interactions have remained unanswered, namely: 1) the primacy of sequence or secondary structure of RNA in determining REV binding, and 2) the roles of oligomerization and effector domains of REV protein in RNA binding. We measured the kinetic and stoichiometric parameters of this interaction in vitro using a Surface Plasmon Resonance (SPR) biosensor. For this purpose, we developed a novel method of immobilizing RNA on the sensor surface that may also be of general use for detecting novel nucleic acid binding proteins. Our results showed that the primary Rev binding site is a core stem-loop RNA molecule of 30 nucleotides (nt) which bound Rev at a 1:1 ratio, while the 244 nt full length RRE bound four Rev monomers. At high Rev concentrations, additional binding of Rev to the 244 nt RRE was observed with Rev:RRE ratios of more than 10:1. However, even RRE mutants that lacked the core binding site and were inactive in vivo bound Rev at these high concentrations. Therefore, in vitro, the stoichiometric ratio of Rev:RRE for specific binding is close to 4:1. Full length RRE displayed a binding affinity for Rev calculated to be approximately 100 pico-molar, but the affinity of Rev for the core RNA was estimated to be 10 pico-molar. This difference was due to the contribution of low affinity Rev binding sites on the full length RRE to calculations of total binding affinity. The ability to evaluate in a step-wise manner the contributions of binding sites of differing affinity by SPR analysis is a definite improvement over other classical methods of nucleic acid : protein binding analysis that tend to average kinetic contributions of the different binding sites under pseudo- steady-state conditions. Mathematical fitting of the primary sensograms suggested co-operativity of Rev binding, probably mediated by the Rev oligomerization domains. Deletions at the C-terminus of Rev had no effect on binding specificity, stoichiometry or kinetics, but deletions of as few as 11 N-terminal amino acids significantly reduced Rev binding specificity. Finally, we demonstrated that this method could be used to rapidly and conveniently evaluate the potential of different compounds, in this case, aminoglycoside antibiotics, to inhibit the Rev:RRE interaction.Among the accessory proteins encoded by HIV-1, the 27 kDal membrane associated myristoylated Nef has proved to be enigmatic. However, there is reasonable agreement in the literature on the potential roles of Nef in modulating certain cellular receptors. In this report, we have tried to address specific properties of Nef in this area by mechanistic studies of Nef effects on CD4 and major histocompatibility complex Class I receptor (MHC-I) and chemokine receptors.We have observed in the past that Nef downregulated CD4 by a bi-modal mechanism. Enhanced endocytosis of CD4 is the preponderant effect when Nef was expressed acutely in T cell lines. However, when Nef and CD4 were transiently co-expressed, Nef induced defect(s) in the early steps of CD4 synthesis and transport in addition to the accelerated endocytosis of the CD4 receptor. The cytoplasmic CD4 defect may have been due to interference with the intracellular vesicular traffic of certain nascent proteins in Nef expressing cells. The ensuing delay in the recruitment of selected membrane proteins such as CD4 into these vesicles may lead to their premature degradation. Two distinct sets of chemokine receptors have been shown to be needed for HIV entry into CD4 positive cells. They are the CCR5 and CXCR4 receptors that are used by the macrophage (M)-tropic and T cell (T)- tropic strains of HIV respectively. We found that Nef proteins of HIV and SIV differentially modulated CCR5 and CXCR4 with potential implications for tropism switching during natural infection.Nef induced a 8-30 fold and a 4-20 fold down-modulation of cell surface CD4 and CCR5 respectively. The functional consequence of this effect was confirmed by ligand CCR5 ligand (RANTES or MIP-1beta) induced receptor phosphorylation and calcium flux. However, even with the maximal levels of Nef effect, there was no significant effect on the virus entry of M- tropic strains of HIV-1. Nef effect was dose dependent, and certain HIV nef alleles and SIV Nef were quite potent in this respect. CCR5 mutants with truncated tails were downregulated by Nef, if they are expressed on the cell surface. CCR5 tail mutants that were devoid of phosphorylation and signaling phenotypes were still sensitive to Nef effect. In contrast to the effect on CCR5, SIV Nef induced downregulation of CXCR4 resulted in corresponding suppression of virus entry by the T-tropic strains of HIV-1. These results led us to conclude that the threshold of CCR5 for HIV entry is lower than that of CXCR4 and propose that Nef may act as a tropism switching factor during natural infection.MHC-I expression levels and usage status are important in AIDS pathogenesis since perturbation of antigen presentation by infected cells may allow them to escape T killer cell surveillance that is a vital immune mechanism against virus infection. We undertook an exhaustive analysis of potential Nef effects on MHC-I. Nef effects were highly variable. On some cells, notably, three HeLa cell clones and two different HEK 293-T cells, cell surface MHC-I expression was not altered by Nef expression. With some HEK 293 parental cell lines, Nef expression resulted in the loss of staining with some MHC-I MAbs but not with others. Similar situation prevailed with U937 macrophage cell lines. Jurkat cells demonstrated marked loss of staining with many different MHC-I MAbs. With these cell lines, there was also significant loss of staining for beta-MCG. Two different HIV Nef alleles, but not SIV Mac 239 Nef behaved similarly. Where the Nef effect was quite impressive for cell surface expression of MHC-I, there was no evidence for increased turnover of HLA-I demonstrable by pulse-chase labeling or receptor internalization experiments.Nef has also been implicated in the biochemical pathways leading up to cellular proliferation and/or apoptosis. Whether the Nef interferes specific receptor mediated cell signaling cascade or downstream events regulating cell cycle are being investigated.